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All-trans-retinoic acid (RA) plays a crucial role in survival and differentiation of neurons. For elucidating signaling mechanisms involved in RA-induced neuronal differentiation, we have selected SH-SY5Y cells, which are an established in vitro cell model for studying RA signaling. Here we report that RA-induced neuronal differentiation of SH-SY5Y cells is coupled with increased expression/activation of TGase and in vivo transamidation and activation of RhoA. In addition, RA promotes formation of stress fibers and focal adhesion complexes, and activation of ERK1/2, JNK1, and p38alpha/beta/gamma MAP kinases. Using C-3 exoenzyme (RhoA inhibitor) or monodansylcadaverine (TGase inhibitor), we show that transamidated RhoA regulates cytoskeletal rearrangement and activation of ERK1/2 and p38gamma MAP kinases. Further, by using stable SH-SY5Y cell lines (overexpressing wild-type, C277S mutant, and antisense TGase), we demonstrate that transglutaminase activity is required for activation of RhoA, ERK1/2, JNK1, and p38gamma MAP kinases. Activated MAP kinases differentially regulate RA-induced neurite outgrowth and neuronal marker expression. The results of our studies suggest a novel mechanism of RA signaling, which involves activation of TGase and transamidation of RhoA. RA-induced activation of TGase is proposed to induce multiple signaling pathways that regulate neuronal differentiation.  相似文献   

3.
Type III secretion systems are used by several pathogens to translocate effector proteins into host cells. Yersinia pseudotuberculosis delivers several Yop effectors (e.g. YopH, YopE and YopJ) to counteract signalling responses during infection. YopB, YopD and LcrV are components of the translocation machinery. Here, we demonstrate that a type III translocation protein stimulates proinflammatory signalling in host cells, and that multiple effector Yops counteract this response. To examine proinflammatory signalling by the type III translocation machinery, HeLa cells infected with wild-type or Yop-Y. pseudotuberculosis strains were assayed for interleukin (IL)-8 production. HeLa cells infected with a YopEHJ- triple mutant released significantly more IL-8 than HeLa cells infected with isogenic wild-type, YopE-, YopH- or YopJ- bacteria. Complementation analysis demonstrated that YopE, YopH or YopJ are sufficient to counteract IL-8 production. IL-8 production required YopB, but did not require YopD, pore formation or invasin-mediated adhesion. In addition, YopB was required for activation of nuclear factor kappa B, the mitogen-activated protein kinases ERK and JNK and the small GTPase Ras in HeLa cells infected with the YopEHJ- mutant. We conclude that interaction of the Yersinia type III translocator factor YopB with the host cell triggers a proinflammatory signalling response that is counteracted by multiple effectors in host cells.  相似文献   

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The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.  相似文献   

6.
Legionella pneumophila enhC - mutants were originally identified as being defective for uptake into host cells. In this work, we found that the absence of EnhC resulted in defective intracellular growth when dissemination of intracellular bacteria to neighbouring cells was expected to occur. No such defect was observed during growth within the amoeba Dictyostelium discoideum. Culture supernatants containing the secreted products of infected macrophages added to host cells restricted the growth of the Δ enhC strain, while tumour necrosis factor α (TNF-α), at concentrations similar to those found in macrophage culture supernatants, could reproduce the growth restriction exerted by culture supernatants on L. pneumophila Δ enhC . The absence of EnhC also caused defective trafficking of the Legionella- containing vacuole in TNF-α-treated macrophages. EnhC was shown to be an envelope-associated protein largely localized to the periplasm, with its expression induced in post-exponential phase, as is true for many virulence-associated proteins. Furthermore, the absence of EnhC appeared to affect survival under stress conditions, as the Δ enhC mutant was more susceptible to H2O2 treatment than the wild-type strain. EnhC therefore is a unique virulence factor that is required for growth specifically when macrophages have heightened potential to restrict microbial replication.  相似文献   

7.
Mitogen-activated protein (MAP) kinases play a pivotal role in the macrophages in the production of proinflammatory cytokines triggered by lipopolysaccharides. However, their function in the responses of macrophages to Gram-positive bacteria is poorly understood. Even less is known about the attenuation of MAP kinase signaling in macrophages exposed to Gram-positive bacteria. In the present study, we have investigated the regulation of MAP kinases and the role of MAP kinase phosphatase (MKP)-1 in the production of pro-inflammatory cytokines using murine RAW264.7 and primary peritoneal macrophages after peptidoglycan stimulation. Treatment of macrophages with peptidoglycan resulted in a transient activation of JNK, p38, and extracellular signal-regulated kinase. Most interestingly, MKP-1 expression was potently induced by peptidoglycan, and this induction was concurrent with MAP kinase dephosphorylation. Triptolide, a diterpenoid triepoxide, potently blocked the induction of MKP-1 by peptidoglycan and prolonged the activation of JNK and p38. Overexpression of MKP-1 substantially attenuated the production of tumor necrosis factor (TNF)-alpha induced by peptidoglycan, whereas knockdown of MKP-1 by small interfering RNA substantially increased the production of both TNF-alpha and interleukin-1 beta. Finally, we found that in primary murine peritoneal macrophages, MKP-1 induction following peptidoglycan stimulation also coincided with inactivation of JNK and p38. Blockade of MKP-1 induction resulted in a sustained activation of both JNK and p38 in primary macrophages. Our results reveal that MKP-1 critically regulates the expression of TNF-alpha and interleukin-1 beta in RAW264.7 cells and further suggest a central role for this phosphatase in controlling the inflammatory responses of primary macrophages to Gram-positive bacterial infection.  相似文献   

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Mitogen-activated protein (MAP) kinases are critical mediators of innate immune responses. In response to lipopolysaccharide (LPS), MAP kinases are rapidly activated and play an important role in the production of proinflammatory cytokines. Although a number of MAP kinase phosphatases (MKPs) have been identified, their roles in the control of cytokine production have not been well defined. In the present report, we investigated the role of MKP-1 in alveolar macrophages stimulated with LPS. We found that LPS triggered transient activation of three MAP kinase subfamilies, ERK, JNK, and p38, in both immortalized and primary murine alveolar macrophages. MKP-1 was rapidly induced by LPS, and its induction correlated with the dephosphorylation of these MAP kinases. Blocking MKP-1 with triptolide prolonged the activities of both JNK and p38 in immortalized alveolar macrophages. Stimulation of primary alveolar macrophages isolated from MKP-1-deficient mice with LPS resulted in a prolonged p38 phosphorylation compared with wild type alveolar macrophages. Accordingly, these MKP-1-deficient alveolar macrophages also mounted a more robust and rapid tumor necrosis factor alpha production than their wild type counterparts. Adenovirus-mediated MKP-1 overexpression significantly attenuated tumor necrosis factor alpha production in immortalized alveolar macrophages. Finally, MKP-1 was induced by a group of corticosteroids frequently prescribed for the treatment of inflammatory lung diseases, and the anti-inflammatory potencies of these drugs closely correlated with their abilities to induce MKP-1. Our studies indicated that MKP-1 plays an important role in dampening the inflammatory responses of alveolar macrophages. We speculate that MKP-1 may represent a novel target for therapeutic intervention of inflammatory lung diseases.  相似文献   

9.
Mitogen-activated protein (MAP) kinases have been implicated as important mediators of the inflammatory response. Here we report that c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAP kinase activities are reprogrammed during the IL-6 induced macrophage-like differentiation of the murine myeloid M1 cell line. Moreover, p38 inhibition upregulates JNK and ERK activity in M1 cells and in thioglycollate-elicited peritoneal exudate macrophages. IL-6-induced M1 differentiation also induces expression of the anti-inflammatory cytokine IL-10, and p38 inhibition potentiates this increase in IL-10 expression in an ERK-dependent manner. Thus, we speculate that during inflammatory conditions in vivo macrophage p38 may regulate JNK and ERK activity and inhibit IL-10 expression. These data highlight the importance of p38 in the molecular mechanisms of macrophage function.  相似文献   

10.
Yersinia pseudotuberculosis binds to beta1 integrin receptors, and uses the type III secretion proteins YopB and YopD to introduce pores and to translocate Yop effectors directly into host cells. Y. pseudotuberculosis lacking effectors that inhibit Rho GTPases, YopE and YopT, have high pore forming activity. Here, we present evidence that Y. pseudotuberculosis selectively modulates Rho activity to induce cellular changes that control pore formation and effector translocation. Inhibition of actin polymerization decreased pore formation and YopE translocation in HeLa cells infected with Y. pseudotuberculosis. Inactivation of Rho, Rac, and Cdc42 by treatment with Clostridium difficile toxin B inhibited pore formation and YopE translocation in infected HeLa cells. Expression of a dominant negative form of Rac did not reduce the uptake of membrane impermeable dyes in HeLa cells infected with a pore forming strain YopEHJT(-). Similarly, the Rac inhibitor NSC23766 did not decrease pore formation or translocation, although it efficiently hindered Rac-dependent bacterial uptake. In contrast, C. botulinum C3 potently reduced pore formation and translocation, implicating Rho A, B, and/or C in the control of the Yop delivery. An invasin mutant (Y. pseudotuberculosis invD911E) that binds to beta1 integrins, but inefficiently transduces signals through the receptors, was defective for YopE translocation. Interfering with the beta1 integrin signaling pathway, by inhibiting Src kinase activity, negatively affected YopE translocation. Additionally, Y. pseudotuberculosis infection activated Rho by a mechanism that was dependent on YopB and on high affinity bacteria interaction with beta1 integrin receptors. We propose that Rho activation, mediated by signals triggered by the YopB/YopD translocon and from engagement of beta1 integrin receptors, stimulates actin polymerization and activates the translocation process, and that once the Yops are translocated, the action of YopE or YopT terminate delivery of Yops and prevents pore formation.  相似文献   

11.
Type III secretion-dependent translocation of Yop (Yersinia outer proteins) effector proteins into host cells is an essential virulence mechanism common to the pathogenic Yersinia species. One unique feature of this mechanism is the polarized secretion of Yops, i.e. Yops are only secreted at the site of contact with the host cell and not to the surrounding medium. In vitro, secretion occurs in Ca2+-depleted media, a condition believed to somehow mimic cell contact. Three proteins, YopN, LcrG and TyeA have been suggested to control secretion and mutating any of these genes results in constitutive secretion. In addition, in Y. enterocolitica TyeA has been implied to be specifically required for delivery of a subset of Yop effectors into infected cells. In this work we have investigated the role of TyeA in secretion and translocation of Yop effectors by Y. pseudotuberculosis. An in frame deletion mutant of tyeA was found to be temperature-sensitive for growth and this phenotype correlated to a lowered expression of the negative regulatory element LcrQ. In medium containing Ca2+, Yop expression was somewhat elevated compared to the wild-type strain and low levels of Yop secretion was also seen. Somewhat surprisingly, expression and secretion of Yops was lower than for the wild-type strain when the tyeA mutant was grown in Ca2+-depleted medium. Translocation of YopE, YopH, YopJ and YopM into infected HeLa cells was significantly lower in comparison with the isogenic wild-type strain and Yop proteins could also be recovered in the tissue culture medium. This indicated that the tyeA mutant had lost the ability to translocate Yop proteins by a polarized mechanism. In order to exclude that the defect in translocation seen in the tyeA mutant was a result of lowered expression/secretion of Yops, a double lcrQ/tyeA mutant was constructed. This strain was de-repressed for Yop expression and secretion but was still impaired for translocation of both YopE and YopM. In addition, the low level of YopE translocation in the tyeA mutant was independent of the YopE chaperone YerA/SycE. TyeA was found to localize to the cytoplasm of the bacterium and we were unable to find any evidence that TyeA was secreted or surface located. From our studies in Y. pseudotuberculosis we conclude that TyeA is involved in regulation of Yop expression and required for polarized delivery of Yop effectors in general and is not as suggested in Y. enterocolitica directly required for translocation of a subset of Yop effectors.  相似文献   

12.
The Bcl-2 family member Bad is a pro-apoptotic protein, and phosphorylation of Bad by cytokines and growth factors promotes cell survival in many cell types. Induction of apoptosis by UV radiation is well documented. However, little is known about UV activation of cell survival pathways. Here, we demonstrate that UVB induces Bad phosphorylation at serine 112 in JNK1, RSK2, and MSK1-dependent pathways. Inhibition of mitogen-activated protein (MAP) kinases including ERKs, JNKs, and p38 kinase by the use of their respective dominant negative mutant or a specific inhibitor for MEK1 or p38 kinase, PD98059 or SB202190, resulted in abrogation of UVB-induced phosphorylation of Bad at serine 112. Incubation of active MAP kinase members with Bad protein showed serine 112 phosphorylation of Bad by JNK1 only. However, activated RSK2 and MSK1, downstream kinases of ERKs and p38 kinase, respectively, also phosphorylated Bad at serine 112 in vitro. Cells from a Coffin-Lowry syndrome patient (deficient in RSK2) or expressing an N-terminal or C-terminal kinase-dead mutant of MSK1 were defective for UVB-induced serine 112 phosphorylation of Bad. Furthermore, MAP kinase pathway-dependent serine 112 phosphorylation was shown to be required for dissociation of Bad from Bcl-X(L). These data illustrated that UVB-induced phosphorylation of Bad at serine 112 was mediated through MAP kinase signaling pathways in which JNK1, RSK2, and MSK1 served as direct mediators.  相似文献   

13.
Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.  相似文献   

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Recently, it has been found that overproduction of IL-12 can be dangerous to the host as it is involved in the pathogenesis of a number of autoimmune inflammatory diseases such as multiple sclerosis. It is composed of two different subunits – p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the CNS of patients with Multiple Sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. The present study was undertaken to explore the role of p40 in the expression of TNF-α in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-α in BV-2 microglial cells. This induction of TNF-α production was accompanied by an induction of TNF-α mRNA. In addition to BV-2 glial cells, p70, p402 and p40 also induced the production of TNF-α in mouse primary microglia and peritoneal macrophages. Since the activation of both NF-κB and C/EBPb is important for the expression of TNF-α in microglial cells, we investigated the effect of p40 on the activation of NF-κB as well as C/EBPb. Activation of NF-κB as well as C/EBPb by p40 and inhibition of p40-induced expression of TNF-α by Dp65, a dominant-negative mutant of p65, and DC/EBPb, a dominant-negative mutant of C/EBPb, suggests that p40 induces the expression of TNF-α through the activation of NF-κB and C/EBPb. This study delineates a novel role of IL-12 p40 in inducing the expression of TNF-α in microglial cells which may participate in the pathogenesis of neuroinflammatory diseases.
Acknowledgements:   This study was supported by NIH grants (NS39940 and AG19487).  相似文献   

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Viboud GI  Bliska JB 《The EMBO journal》2001,20(19):5373-5382
The bacterial pathogen Yersinia pseudotuberculosis uses type III secretion machinery to translocate Yop effector proteins through host cell plasma membranes. A current model suggests that a type III translocation channel is inserted into the plasma membrane, and if Yops are not present to fill the channel, the channel will form a pore. We examined the possibility that Yops act within the host cell to prevent pore formation. Yop- mutants of Y.pseudotuberculosis were assayed for pore-forming activity in HeLa cells. A YopE- mutant exhibited high levels of pore-forming activity. The GTPase-downregulating function of YopE was required to prevent pore formation. YopE+ bacteria had increased pore-forming activity when HeLa cells expressed activated Rho GTPases. Pore formation by YopE- bacteria required actin polymerization. F-actin was concentrated at sites of contact between HeLa cells and YopE- bacteria. The data suggest that localized actin polymerization, triggered by the type III machinery, results in pore formation in cells infected with YopE- bacteria. Thus, translocated YopE inhibits actin polymerization to prevent membane damage to cells infected with wild-type bacteria.  相似文献   

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Mycobacterium avium-intracellulare (MAI) is a ubiquitous environmental pathogen that causes disseminated infection in immunocompromised patients, such as those with human immunodeficiency virus, interleukin-12 deficiency, or interferon-γ receptor mutation. Colony morphotypes are associated with MAI pathogenicity. Our previous studies have reported that smooth-transparent (SmT) morphotypes are more virulent and induce less cytokine (interleukin-1β and tumor necrosis factor-α) production by human monocytes than the smooth-domed (SmD) morphotypes. Mitogen-activated protein (MAP) kinases such as extracellular-regulated kinase (ERK) are activated by the phagocytosis of particle antigens in macrophages, and this ERK activation subsequently influences cytokine expression and the control of intracellular pathogen growth. The influence of MAP kinase activation on MAI replication in human monocytes was examined. Peripheral blood monocytes isolated from healthy subjects by Ficoll-Hypaque sedimentation were infected with virulent SmT or avirulent SmD MAI without or with MAP kinase inhibitors. MAP kinase activities were determined by in vitro kinase assay, intracellular MAI growth by CFU assay, and cytokines by enzyme-linked immunosorbent assay. MAI infection induced ERK and p38 activation. Inhibition of ERK by PD98059, but not p38, significantly increased intracellular MAI growth. Tumor necrosis factor-α release and interleukin-1β production in response to MAI were reduced by MAP kinase inhibition. p38 inhibition tended to reduce cytokine production more substantially. These data suggest that ERK activation limits intra-monocytic MAI replication and enhances monocytic cytokine release, whereas p38 activation influences only cytokine release. The effect of MAP kinases on MAI growth might thus be mediated by the modulation of cytokine production.  相似文献   

20.
During infection of cultured epithelial cells, surface-located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non-polar yopB mutant strain displays a wild-type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage-like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia-induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB-dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell-to-cell transfer of Yop effector proteins.  相似文献   

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